title: Next generation tools for high-throughput promoter and expression analysis employing single-copy knock-ins at the Hprt1 locus.
abstract: We have engineered a set of useful tools that facilitate targeted single copy knock-in (KI) at the hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) locus. We employed fine scale mapping to delineate the precise breakpoint location at the Hprt1(b-m3) locus allowing allele specific PCR assays to be established. Our suite of tools contains four targeting expression vectors and a complementing series of embryonic stem cell lines. Two of these vectors encode enhanced green fluorescent protein (EGFP) driven by the human cytomegalovirus immediate-early enhancer/modified chicken beta-actin (CAG) promoter, whereas the other two permit flexible combinations of a chosen promoter combined with a reporter and/or gene of choice. We have validated our tools as part of the Pleiades Promoter Project (http://www.pleiades.org), with the generation of brain-specific EGFP positive germline mouse strains.
authors.affiliation: Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.
authors.forename: G S
authors.initials: GS
authors.lastname: Yang
authors.forename: K G
authors.initials: KG
authors.lastname: Banks
authors.forename: R J
authors.initials: RJ
authors.lastname: Bonaguro
authors.forename: G
authors.initials: G
authors.lastname: Wilson
authors.forename: L
authors.initials: L
authors.lastname: Dreolini
authors.forename: C N
authors.initials: CN
authors.lastname: de Leeuw
authors.forename: L
authors.initials: L
authors.lastname: Liu
authors.forename: D J
authors.initials: DJ
authors.lastname: Swanson
authors.forename: D
authors.initials: D
authors.lastname: Goldowitz
authors.forename: R A
authors.initials: RA
authors.lastname: Holt
authors.forename: E M
authors.initials: EM
authors.lastname: Simpson
mesh_terms: D000818:Animals; D001483:Base Sequence; D003587:Cytomegalovirus / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D055879:Gene Knock-In Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015203:Reproducibility of Results; D016415:Sequence Alignment; D017384:Sequence Deletion
chemical_list: D007041:Hypoxanthine Phosphoribosyltransferase